Types A, B, E and F botulinum toxins are all of mol. wt. close to 150,000. Toxin purified from nonproteolytic culture has only single chain (unnicked) molecules while those from proteolytic cultures have either all dichain (nicked) molecules of disulfide-linked subunits of 100,000 (H chain) and 50,000 (L chain) mol. wt. or are mixtures of unnicked and nicked molecules. The evidence needed to confirm that all toxin types are synthesized as unnicked molecules is isolation of such molecules from type A cultures. Such molecular form should be present in extracts of cells. Protease(s) of proteolytic cultures converting unnicked to nicked molecular form can be identify by its ability to activate type E toxin to higher specific toxicity. Such enzymes will be isolated and characterized. Quantitating the amounts of toxin that are nicked and the increases in toxicity will be used to examine probability that toxicity increase of E toxin during trypsinization is due to formation of nicked molecules. End-group amino acid analyses will be used to determine if complete activation needs scission of peptide bonds other than one creating the dichain molecule. Separation of H and L chains without protein denaturant will be studied so that their individual antigenicity and toxicity can be ascertained. Some of the results can be used in amino acid sequencing of the botulinum toxins.